Bone remodeling is the dynamic process by which tissue mass and skeletal architecture are maintained. The process is a balance between bone resorption and bone formation, with two cell types, the osteoclast and osteoblast, thought to be the major players. Osteoblasts synthesize and deposit new bone into cavities that are excavated by osteoclasts. The activities of osteoblasts and osteoclasts are regulated by many factors, systemic and local, including growth factors.
Many of the proteins that influence the proliferation, differentiation, and activity of osteoblasts, osteoclasts, and their precursors also affect these processes in chondrocytes, the cells responsible for cartilage formation (chondrogenesis). These proteins include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-xcex2), bone morphogenetic proteins (BMPs), and cartilage-derived growth factor (CDGF).
The exact mode by which PDGF affects the growth of osteoblasts is not yet clearly understood, however, this growth factor is generally believed to play a key role in the regulation of both normal skeletal remodeling and fracture repair. Biologically active PDGF is found as a homodimer or a heterodimer of the component A and B chains. In vitro studies have shown PDGF to be mitogenic for osteoblasts (Abdennagy et al., Cell Biol. Internat. Rep. 16(3):235-247, 1992). Mitogenic activity as well as chemotactic activities associated with PDGF have been demonstrated when the growth factor is added to normal osteoblast-like cells (Tuskamota et al., Biochem. Biophys. Res. Comm. 175(3):745-747, 1991) and primary osteoblast cultures (Centrella et al. Endocrinol. 125(1):13-19, 1989). Recent studies have demonstrated that the osteoblast produces the AA isoform of PDGF (Zhang et al., Am. J. Physiol. 261:c348-c354, 1991).
PDGF has been shown to be useful for promoting the repair of both soft and hard tissues. For example, PDGF has been shown to promote the regeneration of bone and ligament in patients suffering from periodontal disease (Howell et al., J. Periodontol. 68:1186-1193, 1997). As disclosed in U.S. Pat. No. 5,533,836, PDGF stimulates the growth of osteoblasts, and this activity is enhanced in the presence of vitamin D. PDGF has also been shown to promote the healing of gastrointestinal ulcers (U.S. Pat. No. 5,234,908) and dermal ulcers (Robson et al., Lancet 339:23-25, 1992; Steed et al., J. Vasc. Surg. 21:71-81, 1995). The use of PDGF for stimulating chondrocyte proliferation and regenerating cartilage is disclosed in U.S. Pat. No. 6,001,352.
A PDGF homolog known as xe2x80x9czvegf3xe2x80x9d was recently identified (U.S. patent application Ser. No. 09/457,066). This protein has also been designated xe2x80x9cVEGF-Rxe2x80x9d (WIPO Publication WO 99/37671). Zvegf3/VEGF-R is a multi-domain protein with significant homology to the PDGF/VEGF family of growth factors. WO 99/37671 discloses that VEGF-R is an angiogenic factor.
Despite the increasing knowledge of the role of growth factors in tissue growth and repair, there remains a need in the art for materials and methods for promoting the growth of bone, ligament, and cartilage. There also remains a need the art for materials and methods for modulating the proliferation and differentiation of cells in vitro and in vivo.
The present invention provides a method for promoting growth of bone, ligament, or cartilage in a mammal comprising administering to said mammal a composition comprising a pharmacologically effective amount of a dimeric protein comprising residues 235-345 of SEQ ID NO:2 or SEQ ID NO:4 and a pharmaceutically acceptable delivery vehicle. Within certain embodiments of the invention the delivery vehicle is powdered bone, tricalcium phosphate, hydroxyapatite, polymethacrylate, a biodegradable polyester, an aqueous polymeric gel, or a fibrin sealant. Within another embodiment of the invention the composition is locally administered at a site of a bony defect, such as a fracture, bone graft site, implant site, or periodontal pocket. Within another embodiment of the invention, the composition is administered systemically. Within a further embodiment of the invention, the zvegf3 protein is covalently linked to a bone-targetting agent. Within a further embodiment of the invention, the composition is locally administered at a joint. The composition may further comprise a protein selected from the group consisting of insulin-like growth factor 1, platelet-derived growth factor, epidermal growth factor, transforming growth factor-alpha, transforming growth factor-beta, a bone morphogenetic protein, parathyroid hormone, osteoprotegerin, a fibroblast growth factor, and a protein comprising residues 258-370 of SEQ ID NO:5 (a zvegf4 protein). Within another embodiment of the invention, the protein is a homodimer. Within a related embodiment, the protein comprises a first polypeptide chain disulfide bonded to a second polypeptide chain, each of the chains consisting of residues X-345 of SEQ ID NO:2, wherein X is an integer from 226 to 235, inclusive.
The invention also provides a method for promoting growth of bone, ligament, or cartilage in a mammal comprising administering to said mammal a composition comprising a pharmacologically effective amount of a dimeric protein comprising a first polypeptide chain disulfide bonded to a second polypeptide chain, each of the chains comprising of residues 235-345 of SEQ ID NO:2 or SEQ ID NO:4, and a pharmaceutically acceptable delivery vehicle. Within certain embodiments, each of the chains consists of residues X-345 of SEQ ID NO:2, wherein X is an integer from 226 to 235, inclusive. Within other embodiments, each of the chains consists of residues X-345 of SEQ ID NO:2, wherein X is an integer from 15 to 20, inclusive.
The invention also provides a method for promoting proliferation or differentiation of cells comprising culturing the cells in an effective amount of a dimeric protein comprising residues 235-345 of SEQ ID NO:2 or SEQ ID NO:4, wherein the cells are osteoblasts, osteoclasts, chondrocytes, or bone marrow stem cells. Within one embodiment the cells are bone marrow stem cells, and the method comprises harvesting the bone marrow stem cells from a patient prior to culturing. Within other embodiments the method further comprises the step of recovering osteoblasts, osteoclasts, or chrodrocytes from the cultured cells. Within additional embodiments the protein comprises a first polypeptide chain disulfide bonded to a second polypeptide chain, each of the chains consisting of residues X-345 of SEQ ID NO:2, wherein X is an integer from 226 to 235, inclusive.
The invention also provides a method for promoting cartilage growth comprising the steps of (a) culturing chondrocytes ex vivo in the presence of a dimeric protein comprising residues 235-345 of SEQ ID NO:2 or SEQ ID NO:4 under conditions wherein the chondrocytes proliferate, and (b) placing the cultured chondrocytes into a mammal where cartilage is to be grown. Within one embodiment the chondrocytes are placed into the mammal in association with a biodegradable matrix having sufficient porosity to permit cell ingrowth. Within a related embodiment the matrix comprises a protein selected from the group consisting of insulin-like growth factor 1, platelet-derived growth factor, epidermal growth factor, transforming growth factor-alpha, transforming growth factor-beta, a bone morphogenetic protein, parathyroid hormone, a fibroblast growth factor, a protein comprising residues 258-370 of SEQ ID NO:5, and a dimeric protein comprising residues 235-345 of SEQ ID NO:2 or SEQ ID NO:4. Within other embodiments the protein comprises a first polypeptide chain disulfide bonded to a second polypeptide chain, each of the chains consisting of residues X-345 of SEQ ID NO:2, wherein X is an integer from 226 to 235, inclusive.
The invention further provides a method for stimulating proliferation of osteoblasts or chondrocytes in a mammal comprising administering to the mammal a composition comprising a pharmacologically effective amount of a dimeric protein comprising residues 235-345 of SEQ ID NO:2 or SEQ ID NO:4 in combination with a pharmaceutically acceptable delivery vehicle. Within certain embodiments of the invention the delivery vehicle is powdered bone, tricalcium phosphate, hydroxyapatite, polymethacrylate, a biodegradable polyester, an aqueous polymeric gel, or a fibrin sealant. Within another embodiment the composition is locally administered at a site of a bony defect, such as a fracture, bone graft site, implant site, or periodontal pocket. Within another embodiment the composition is administered systemically. Within a further embodiment the zvegf3 protein is covalently linked to a bone-targetting agent. Within an additional embodiment the composition is locally administered at a joint. Within other embodiments the composition further comprises a protein selected from the group consisting of insulin-like growth factor 1, platelet-derived growth factor, epidermal growth factor, transforming growth factor-alpha, transforming growth factor-beta, a bone morphogenetic protein, parathyroid hormone, osteoprotegerin, a fibroblast growth factor, and a protein comprising residues 258-370 of SEQ ID NO:5. Within additional embodiments the protein comprises a first polypeptide chain disulfide bonded to a second polypeptide chain, each of the chains consisting of residues X-345 of SEQ ID NO:2, wherein X is an integer from 226 to 235, inclusive.